Myocardial Ischemia In Rats Research Articles

Effect of atorvastatin on serum oxidative stress and N-terminal brain natriuretic peptide expression in rats

ObjectiveTo investigate the effect of atorvastatin on serum oxidative stress and N-terminal brain natriuretic peptide expression in rats. MethodsA total of 40 healthy male SD rats were randomly divided into the sham group (Group A, n=10, saline 5 mL/d), ischemia-reperfusion group (Group B, n=10, saline 5 mL/d), atorvastatin group (Group C, n=10, atorvastatin 20 mg/kg•d), atorvastatin + N-amino-arginine group (Group D, n=10, atorvastatin 20 mg/kg•d + N-amino arginine 15 mg/kg). Myocardial ischemia-reperfusion rat model was established after 3 days of gavage. N-amino arginine 15 mg/kg was given by tail vein injection 15 min before ischemia. After reperfusion, enzymology indicators such us creatine kinase (CK) and lactate dehydrogenase and the oxidative stress parameters such as nitric oxide (NO), malondialdehyde (MDA) and total superoxide dismutase (TSOD), and n-terminal pro-brain natriuretic peptide (NT-proBNP) expression was detected by immunohistochemistry. ResultsLDH and CK levels of group A were significantly lower than the other three groups, and group B was the highest. There was significant difference between group B and group C (P<0.05), and no significant difference between group B and group D (P>0.05). MDA levels in group B were significantly higher than the other three groups. The lowest was group A, followed by group C, the difference among groups was significantly (P<0.05). TSOD and NO levels in group B was the lowest, the level in group A was the highest, followed by group C, the difference among groups was significant (P<0.05). NT-proBNP level in group B was significantly higher than the other three groups, the lowest was group A, followed by group C, the difference among groups was significant (P<0.05). ConclusionsAtorvastatin has a protective effect on the myocardial injury in the myocardial ischemia and reperfusion rats. It can increase NO synthesis and decrease MDA content, increase serum TSOD activity and the oxidative stress effect, meanwhile protect myocardial cells and reduce myocardial injury.

Acupuncture Promotes Angiogenesis after Myocardial Ischemia through H3K9 Acetylation Regulation at VEGF Gene

BackgroundAcupuncture exerts cardioprotective effects on several types of cardiac injuries, especially myocardial ischemia (MI), but the mechanisms have not yet been well elucidated. Angiogenesis mediated by VEGF gene expression and its modification through histone acetylation has been considered a target in treating myocardial ischemia. This study aims to exam whether modulation of angiogenesis through H3K9 acetylation regulation at VEGF gene is one possible cardioprotective mechanism of acupuncture.ResultsWe generated rat MI models by ligating the left anterior descending coronary artery and applied electroacupuncture (EA) treatment at the Neiguan (PC6) acupoint. Our results showed that acupuncture reversed the S-T segment change, reduced Q-wave area, decreased CK, CK-MB, LDH levels, mitigated myocardial remodeling, and promoted microvessel formation in the MI heart. RNA-seq analysis showed that VEGF-induced angiogenesis signaling was involved in the modulation of EA. Western blot results verified that the protein expressions of VEGF, Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt, were all elevated significantly by EA treatment in the MI heart. Furthermore, increased H3K9 acetylation was also observed according with the VEGF. ChIP assay confirmed that EA treatment could notably stimulate the recruitment of H3K9ace at the VEGF promoter.ConclusionsOur study demonstrates for the first time that acupuncture can effectively up-regulate VEGF expression through H3K9 acetylation modification directly at the VEGF promoter and hence activate VEGF-induced angiogenesis in rat MI models. We employed high throughput sequencing in this study and, for the first time, generated genome-wide gene expression profiles both in the rat MI model and in acupuncture treatment.

Study on PK-PD characteristics of ginsenoside Rg1 and Rb1 in rats with myocardial ischemia following intravenous administration of Shengmai injection

It is the objective of this paper to study pharmacokinetics-pharmacodynamics (PK-PD) characteristics of ginsenoside Rg1 and Rb1 on the effect of inducing nitric oxide (NO) release after intravenous administration of Shengmai injection to rats with myocardial ischemia. The model of myocardial ischemia rats was produced by subcutaneous injection of isoproterenol. The serum samples were collected at different time points after intravenous administration of Shengmai injection to rats with the dose of 10.8 mL x kg(-1). The concentrations of ginsenoside Rg1 and Rb1 in serum were determined, and then the concentration-time curves were drawn. Pharmacokinetic parameters of ginsenoside Rg1 and Rb1 were calculated after the construction of pharmacokinetic models. Meanwhile, NO2- and NO3-, the metabolites of NO, in serum were determined, and then the effect-time curve was drawn. The combined PK-PD model was established based on the theory of effect compartment by Sheiner et al. Then pharmacodynamic parameters were calculated. The results indicated that the pharmacokinetics of ginsenoside Rg1 and Rb1 conformed to a two-compartment model. Ginsenoside Rg1 and Rb1 exhibited quick and slow elimination in rats respectively. The effect of Shengmai injection on inducing NO release did not relate directly with and lagged behind the concentrations of ginsenoside Rg1 and Rb1 in serum. The effect exhibited good correlation with ginsenoside Rg1 and Rb1 levels in effect compartment. The relationship between effect and serum concentration fits Sigmoid-E(max) model. This study successfully established the combined PK-PD model of ginsenoside Rg1 and Rb1 after intravenous administration of Shengmai injection to rats. The model can efficiently predict the concentration and effect of Shengmai injection in vivo.

Puerarin alleviates aggravated sympathoexcitatory response induced by myocardial ischemia via regulating P2X3 receptor in rat superior cervical ganglia

Myocardial ischemia elicits a sympathoexcitatory response characterized by increase in blood pressure and sympathetic nerve activity. Puerarin, a major active ingredient extracted from the traditional Chinese plant medicine Ge-gen, has been widely used in treatment of myocardial and cerebral ischemia. However, little is known about the mechanism. Our study was aimed to explore the effect of puerarin on sympathoexcitatory response induced by myocardial ischemic injury and possible relationship with P2X3 receptor. Our results showed that puerarin alleviated systolic blood pressure and heart rate, and decreased the up-regulated of P2X3 mRNA and protein in SCG of myocardial ischemic rats. The amplitude of ATP-activated currents of SCG neurons was much larger in myocardial ischemic group than that in control group. Puerarin reduced ATP-activated currents in myocardial ischemic group and control group, and the inhibiting effects of puerarin in myocardial ischemic group were stronger than those in control group. Puerarin also significantly inhibited ATP-activated currents in HEK293 cells transfected with P2X3 receptor. These results suggest that puerarin can depress up-sympathoexcitatory response induced by myocardial ischemia via acting on P2X3 receptor in rat SCG to protect myocardium.

Dynamic Alterations in the CaV1.2/CaM/CaMKII Signaling Pathway in the Left Ventricular Myocardium of Ischemic Rat Hearts

Cardiac L-type calcium channel (CaV1.2), calmodulin (CaM), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) form the CaV1.2/CaM/CaMKII signaling pathway, which plays an important role in maintaining intracellular Ca(2+) homeostasis. The roles of CaM and CaMKII in the regulation of CaV1.2 in Ca(2+)-dependent inactivation and facilitation have been reported; however, alterations in this signaling pathway in the heart after myocardial ischemia (MI) had not been well characterized. In this study, we investigated the dynamic changes in CaV1.2, CaM, and CaMKII mRNA and protein expression levels in the left ventricles of the heart following MI in rats. The MI model was induced by ligating the left anterior descending coronary artery; the rats were divided into the following five groups: the 6 h post-MI group (MI-6h), 24 h post-MI group (MI-24h), 1 week post-MI group (MI-1w), 2 weeks post-MI group (MI-2w), and the sham group. The mRNA levels were measured by quantitative real-time polymerase chain reaction and the protein expression was determined by western blotting and immunohistochemistry. There were no observable differences in the CaV1.2 mRNA and protein levels at the early stages of MI, but these levels decreased at MI-2w. Both the mRNA and protein levels of CaM increased at MI-6h, peaked at MI-24h, and then reduced to normal levels at MI-2w. CaMKII mRNA and protein levels decreased at MI-6h and reached their lowest level at MI-24h. Taken together, these data demonstrate that there are dynamic changes in the CaV1.2/CaM/CaMKII signaling pathway following MI injuries, which suggests that different therapeutic regimens should be used at different time points after MI injuries.

Dynamics of interstitial calcium in rat myocardial ischemia reperfusion injury in vivo

Intracellular calcium overload is a key factor for myocardial ischemia reperfusion injury (IR). However, there was no report for interstitial calcium concentration dynamics. We investigated the interstitial calcium dynamics in rat myocardial IR model in vivo. A microdialysis system was involved, and the time delay of the system and recovery time was introduced and tested with a fluids switching method. Twelve SD rats were divided into IR or control group. Myocardial IR was induced by ligating (20 min) then releasing (60 min) the suture underlying left anterior descending branch. Mycrodialyisis probe was implanted into the left ventricular myocardium perfusion area for occlusion. Dialysate samples were collected every 10 min. Dialysate calcium concentration was detected with an atomic absorption spectrophotometer. Recovery time for the microdialysis system was 20 min, and recovery rate was 16%. Dialysate calcium concentration showed no changes during ischemia, descended immediately after reperfusion, reached the lowest level (67% of baseline value) 20 min after reperfusion, then escalated slowly. Recovery time was an important parameter for mycrodialysis technique, and it should not be neglected and needed to be tested. Our data suggest that interstitial calcium concentration in rats with myocardial IR in vivo kept steady in ischemia, descended rapidly at the initial reperfusion, then rebounded slowly. In conclusion, we introduced the concept of recovery time for microdialysis and provided a simple testing method.

The use of antibody modified liposomes loaded with AMO-1 to deliver oligonucleotides to ischemic myocardium for arrhythmia therapy

MicroRNA-1 (miR-1) has been found in cardiac and skeletal tissues. It is overexpressed in ischemic cardiac tissues. Down-regulation of miR-1 could relieve arrhythmogenesis by the anti-miR-1 antisense oligonucleotides (AMO-1). To increase the therapeutic efficiency and inhibit off-target effects of AMO-1, here we explored anti-cardiac troponin I (cTnI) antibody modified liposomes loading with AMO-1 (cT-A-LIP) to deliver the oligonucleotides to ischemic myocardium tissues. Liposomal cytotoxicity was assessed by MTT assay. The targeting abilities to foci were evaluated by in vivo imaging. The uptake and bio-distribution in vitro were observed by live cell station and flow cytometry, respectively. The anti-arrhythmic effects of cT-A-LIP in vivo were evaluated by electrocardiograms (ECG), immunohistochemistry, real-time PCR and patch-clamp recording. Immunohistochemistry showed that cTnI expression had a peak at the third day after myocardial infarction (MI). After cT-LIP administration via tail vein, accumulation of fluorescent trackers in the ischemic foci was significantly increased more than that of LIP. In addition, after cT-A-LIP administration, the ischemic arrhythmias were recovered and ST segment in ECG was elevated nearly back to normal. Compared with MI group, miR-1 expression was significantly down-regulated while Kir2.1 and CX43 protein expression were increased. Patch-clamp recordings showed that cT-A-LIP as well as AMO-1 incubation increased K+ current density in guinea pigs ventricular cardiomyocytes acting on repolarized membrane potential. In conclusion, the cT-A-LIP not only delivered AMO-1 to ischemic myocardium in MI rats, but validated AMO-1 on relieving ischemic arrhythmia by silencing of miR-1 in ischemic myocardium and restoring the depolarized resting membrane potential (RMP) in MI rats.

A pharmacokinetic and pharmacodynamic study of drug–drug interaction between ginsenoside Rg1, ginsenoside Rb1 and schizandrin after intravenous administration to rats

Ethnopharmacological relevanceGinsenoside Rg1, ginsenoside Rb1 and schizandrin are main bioactive components from Panax ginseng and Schisandra chinensis. They have been found in many prescriptions of Traditional Chinese Medicines (TCM) and proven to be effective for prevention and treatment of cardiovascular disease. It is valuable to investigate their pharmacokinetic and pharmacodynamic behavior and potential synergistic effect for better drug development and clinical application. Materials and methodsPharmacokinetic and nitric oxide (NO) release pharmacodynamic drug–drug interactions of ginsenoside Rg1, ginsenoside Rb1 and schisandrin were studied after intravenous administration of each compound with the dose of 10mg/kg and their mixture with the total dose of 10mg/kg to isoproterenol (ISO)-induced myocardial ischemia rats. Drug concentrations in serum were determined using LC–MS method. Nitrite and nitrate (NOx−), the predominant oxidation product of NO in serum was used as an effective marker and quantitated by the method of high-performance liquid chromatography coupled with fluorescence detection (HPLC-FL). The main pharmacokinetic parameters of T1/2β, MRT0−∞, Vd, Cl, and AUC, and the main pharmacodynamic parameters of Cmax, Tmax and AUEC were calculated by non-compartment model. ResultsThe results indicated ginsenoside Rb1 and (or) schisandrin in mixture could significantly postpone the elimination of ginsenoside Rg1 in rat serum. Co-administration of three compounds markedly increased the systemic exposure level of each compound in vivo. Ginsenoside Rg1 and ginsenoside Rb1 had the effect of inducing real-time NO release in rats concentration dependently. Schisandrin had no effect of inducing real-time NO release in this study. The mixture of ginsenoside Rg1, Rb1 and schisandrin administration exhibited synergistic effect of inducing NO release in ISO treated rats. ConclusionsThe result obtained from this study suggested pharmacokinetic and pharmacodynamic drug–drug interactions between ginsenoside Rg1, Rb1 and schisandrin. The study provided valuable information for drug development and clinical application of TCM.

Protective Effects of Berberine on Isoproterenol-Induced Acute Myocardial Ischemia in Rats through Regulating HMGB1-TLR4 Axis.

Berberine, an isoquinoline alkaloid originally isolated from the Chinese herb Coptis chinensis (Huanglian), has been shown to display a wide array of pharmacological activities. The present study was to investigate the effects of berberine against myocardial ischemia produced in rats by isoproterenol. 50 male Sprague-Dawley rats were randomized equally into five groups: a control group, an untreated model group, berberine (30, 60 mg/kg) treatment, or propranolol (30 mg/kg). Rats were treated for 12 days and then given isoproterenol, 85 mg/kg for 2 consecutive days by subcutaneous injection. ST-segment elevation was measured after the last administration. Serum levels of creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured after the rats were sacrificed. The hearts were excised for determining heart weight index, microscopic examination, high mobility group box 1 (HMGB1), toll-like receptor (TLR4), prodeath protein (Bax), antideath protein (Bcl-2), and tumor necrosis factor (TNF-α) protein were determined by western blot. Berberine decreased the ST elevation induced by acute myocardial ischemia, and decreased serum levels of CK-MB, LDH, TNF-α, and IL-6. Berberine increased total superoxide dismutase (T-SOD) activity and decreased malondialdehyde (MDA) content in myocardial tissue. Berberine can regulate HMGB1-TLR4 axis to protect myocardial ischemia.

Naloxone Postconditioning Alleviates Rat Myocardial Ischemia Reperfusion Injury by Inhibiting JNK Activity

To investigate the alteration of c-Jun N-terminal kinase (JNK) activity after myocardial ischemia reperfusion injury (MIRI) and further explore the effect of naloxone postconditioning on MIRI. Forty male Sprague Dawley rats were randomly divided into five groups: sham operation (sham, n=8); ischemia reperfusion (IR, n=8); IR+naloxone 0.5 mg/kg (Nal L, n=8); IR+naloxone 1.0 mg/kg (Nal M, n=8); IR+naloxone 2.0 mg/kg (Nal H, n=8). Pathological changes of myocardial tissue were visualized by HE staining. The expression of p-JNK, and the apoptosis of cardiomyocytes were investigated with Western blotting and the TUNEL assay, respectively. Irregular arrangement and aberrant structure of myocardial fibers, cardiomyocytes with granular or vacuolar degeneration, and inflammatory cells infiltrating the myocardial interstitial regions characterized MIRI in the IR group. Signs of myocardial injury and inflammatory infiltration were less prominent in the Nal-treated groups. The expression of p-JNK in the sham group and in all Nal-treated groups was significantly lower than that in the IR group (p<0.01). The apoptosis index of cardiomyocytes in the IR group was significantly higher than in the sham group (p< 0.01). The apoptosis indices of cardiomyocytes in all Nal-treated groups were significantly reduced to 55.4%, 26.2%, and 27.6%, respectively, of the IR group (p< 0.01). This study revealed that Naloxone postconditioning before reperfusion inhibits p-JNK expression and decreases cell apoptosis, thus alleviating MIRI.

Salvianolic acid A promotes the acceleration of neovascularization in the ischemic rat myocardium and the functions of endothelial progenitor cells

Ethnopharmacological relevanceSalvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely used Chinese herbal medicines in clinical, containing phenolic compounds and potent antioxidant properties. Salvianolic acid A (SAA) is the most potent component of SM. A modern experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by the use of "angiogens", mediators that induce the formation of blood vessels, or angiogenesis. Studies demonstrated that coronary collateral vessels protect ischemic myocardium after coronary obstruction; therefore, we sought to examine whether SAA could stimulate myocardial angiogenesis. Materials and methodsMale Sprague–Dawley rats myocardial infarct (MI) induced by ligation of left anterior descending coronary artery (LAD) were randomly divided into five groups: sham-operated group; LAD occlusion + administration of physiological saline (vehicle treated group); LAD occlusion + administration of different concentrations of SAA (10, 5.0 and 2.5mg/kg/d). Infarct size and capillary density in the infarct region were measured with a previous experimental method. Immunohistological analysis was performed to measure vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) expressions. The secretion of matrix metalloproteinase type X (MMP-9) was evaluated in serum of post-ischemic rats. We also performed the experiments of SAA on rat endothelial progenitor cells (EPCs) numbers and the capacity of migration and vasculargenesis. ResultsSAA potentiated the ischemia-induced neovascularization after 1week post-operation when compared to vehicle treated group. This effect could be attributed to an increased formation of VEGF, VEGFR-2, and MMP-9 as well as the promotion of numbers and functions of EPCs. ConclusionThese findings show that SAA has potent proangiogenic properties by promoting the expression of proangiogenic factors, and the functions of EPCs, indicating that SAA might contribute to the protective effect against coronary disease. Chemical compound studied in this paper is salvianolic acid A (PubChem CID: 5281793)

Dynamics of interstitial calcium in rat myocardial ischemia reperfusion injury in vivo

Intracellular calcium ([Ca++]ic) overload is the key factor for myocardial ischemia reperfusion injury(IR), however there was no report for interstitial Ca++ concentration ([Ca++]ex) dynamics. This research aims to plot the [Ca++]ex dynamics in rat myocardial IR in vivo. A microdialysis system was composed and the time delay of the system, recovery time, was introduced and tested with a fluids switching method. Twelve SD rats were divided into IR or control group. Myocardial IR was induced by ligating (20 min) or releasing (60 min) the suture underlying LAD. Mycrodialyisis probe was implanted into the left ventricular myocardium perfusion area to be occluded. Dialysate samples were collected every 10 min. Blood samples were drawn at the beginning and at the end of the procedures. Dialysate calcium concentration ([Ca++]i) was detected with an atomic absorption spectrophotometer. Serum calcium and cTnT were detected. Results revealed recovery time for the microdialysis system was 20 min, recovery rate was 16%. [Ca++]i showed no changes during ischemia and descended immediately after reperfusion, reached the lowest level at 20 min after reperfusion, then escalated slowly while keeping lower than control with significant difference. There was no difference in serum calcium at the beginning (control vs IR, mmol/L: 2.35±0.31 vs 2.63±0.2, p=0.093) and ending point (control vs IR, mmol/L: 2.38±0.34 vs 2.66±0.15, p=0.095). Serum cTnT of both groups showed no difference at the beginning (control vs IR, ng/mL: 0.47±0.38 vs 0.74±0.46, p=0.287) and rose respectively at the 60 min after reperfusion (control vs IR, ng/mL: 1.62±0.6 vs 4.29±2.22, p=0.031). Given the recovery time and recovery rate, baseline of rat myocardial [Ca++]ex was estimated as 1.26±0.22 mmol/L and the [Ca++]ex was speculated steady in ischemia, descending 33% at the start of reperfusion, then escalating slowly. Thus it can be inferred that recovery time was an important parameter for mycrodialysis technique, which should not be neglected and need to be tested. Our data suggest that [Ca++]ex in rat myocardial IR in vivo kept steady in ischemia, descended rapidly at the initial reperfusion then rebounded slowly. Key words: Myocardium, ischemia, reperfusion, calcium, microdialysis.

The Interaction of Mast Cells and Cardiac Sympathetic Nerves During Acupuncture in Acute Myocardial Ischemia Rats

Abstract Background: Acupuncture has been shown to regulate visceral functions. However, the specific mechanism remains unknown. Objective: This study attempted to investigate cardiac sympathetic nerve activity response to acupuncture and its activation effect on mast cells in the acupoint area. Materials and Methods: A rat model of acute myocardial ischemia (AMI) was prepared by ligating the left anterior descending artery branch. After dissecting a branch of the cardiac sympathetic nerves, the left Neiguan point (PC 6) was stimulated with acupuncture. Then, the skin tissues of acupoint area were sampled and stained with neutral red after killing the rats. Main Outcome Measures: Nerve discharges and electrocardiograph readings were recorded during acupuncture. Heart rate variability (HRV) and the power spectrum of nerve discharges were used as markers for analysis. Ratios of degranulated to total mast cells were also calculated. Results: AMI caused an increase in the ratio of low frequency/high frequency...

Metabonomic analysis of Allium macrostemon Bunge as a treatment for acute myocardial ischemia in rats

Myocardial ischemia (MI) refers to a pathological state of the heart caused by reduced cardiac blood perfusion, which leads to a decreased oxygen supply in the heart and an abnormal myocardial energy metabolism. Acute myocardial ischemia (AMI) has posed a significant health risk for humans. Allium macrostemon Bunge (AMB), a popular traditional Chinese medicine, is used for MI treatment. The therapeutic effects of AMB were assessed and the detailed mechanisms of AMB for AMI treatment were investigated. We characterized the metabonomic variations in rats from the sham surgery, AMI, and AMB-pretreated AMI groups through a combination of nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analysis. Thirty-five metabolites including carbohydrates, a range of amino acids, and organic acids were detected. The 1H NMR spectra of the rat serum were analyzed using the principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA). Results showed that AMI induced some physiological changes in rats and also led to metabolic disorders related to glycolysis promotion, amino acid metabolism disruption, and other metabolite metabolism perturbation. AMB pretreatment reduced the AMI injury and maintained metabolic balance, possibly by limiting the change in energy metabolism and regulating amino acid metabolism. These findings provide a comprehensive insight on the metabolic response of AMI rats to AMB pretreatment and are important for the use of AMB for AMI therapy.

Protection of H9c2 rat cardiomyoblasts against oxidative insults by total paeony glucosides from Radix Paeoniae Rubrae

Total paeony glucosides (TPG) extracted from the roots of Radix Paeoniae Rubrae, have been approved for the therapy of rheumatoid arthritis by the State Food and Drug Administration. We previously demonstrated the myocardial protective effects of TPG in both isoprenaline-induced myocardial ischemia rat and acute myocardial infarction rat. However, the underlying mechanism of TPG effect in cardiomyocytes remains to be investigated. The aims of this study were to elucidate the effect of TPG on the activities of antioxidant defense targets and the bioenergetic system in rat cardiomyocytes. The changes of viability, antioxidant defense system activities, protein contents, and mitochondrial functions in tert-butyl hydroperoxide challenged H9c2 rat cardiomyoblasts were evaluated. The results suggest that TPG ameliorated cardiomyoblast dysfunction by preserving antioxidant defense and bioenergetic system.

Downregulation of miR-151-5p Contributes to Increased Susceptibility to Arrhythmogenesis during Myocardial Infarction with Estrogen Deprivation

Estrogen deficiency is associated with increased incidence of cardiovascular diseases. But merely estrogen supplementary treatment can induce many severe complications such as breast cancer. The present study was designed to elucidate molecular mechanisms underlying increased susceptibility of arrhythmogenesis during myocardial infarction with estrogen deprivation, which provides us a new target to cure cardiac disease accompanied with estrogen deprivation. We successfully established a rat model of myocardial ischemia (MI) accompanied with estrogen deprivation by coronary artery ligation and ovariectomy (OVX). Vulnerability and mortality of ventricular arrhythmias increased in estrogen deficiency rats compared to non estrogen deficiency rats when suffered MI, which was associated with down-regulation of microRNA-151-5p (miR-151-5p). Luciferase Reporter Assay demonstrated that miR-151-5p can bind to the 3′-UTR of FXYD1 (coding gene of phospholemman, PLM) and inhibit its expression. We found that the expression of PLM was increased in (OVX+MI) group compared with MI group. More changes such as down-regulation of Kir2.1/IK1, calcium overload had emerged in (OVX+MI) group compared to MI group merely. Transfection of miR-151-5p into primary cultured myocytes decreased PLM levels and [Ca2+]i, however, increased Kir2.1 levels. These effects were abolished by the antisense oligonucleotides against miR-151-5p. Co-immunoprecipitation and immunofluorescent experiments confirmed the co-localization between Kir2.1 and PLM in rat ventricular tissue. We conclude that the increased ventricular arrhythmias vulnerability in response to acute myocardial ischemia in rat is critically dependent upon down-regulation of miR-151-5p. These findings support the proposal that miR-151-5p could be a potential therapeutic target for the prevention of ischemic arrhythmias in the subjects with estrogen deficiency.

Protective effects of cinnamic acid and cinnamic aldehyde on isoproterenol-induced acute myocardial ischemia in rats

Ethnopharmacological relevanceCinnamomum cassia is a well-known traditional Chinese herb that is widely used for the treatment of ischemic heart disease (IHD). It has favorable effects, but its mechanism is not clear. To investigate the effects of cinnamic aldehyde (CA) and cinnamic acid (CD) isolated from Cinnamomum cassia against myocardial ischemia produced in rats by isoproterenol (ISO). Materials and methodsNinety male Sprague-Dawley rats were randomized equally to nine groups: a control group, an untreated model group, CA (22.5, 45, 90mg/kg) or CD (37.5, 75, 150mg/kg) treatment, or propranolol (30mg/kg). Rats were treated for 14 days and then given ISO, 4mg/kg for 2 consecutive days by subcutaneous injection. ST-segment elevation was measured after the last administration. Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO), and blood rheology were measured after the rats were sacrificed. The hearts were excised for determining heart weight index, microscopic examination, superoxide dismutase (SOD) and malondialdehyde (MDA) measurements. ResultsCA and CD decreased the ST elevation induced by acute myocardial ischemia, decreased serum levels of CK-MB, LDH, TNF-α and IL-6, and increased serum NO activity. CA and CD increased SOD activity and decreased MDA content in myocardial tissue. ConclusionCA and CD were cardioprotective in a rat model of ischemic myocardial injury. The protection was attributable to anti-oxidative and anti-inflammatory properties, as well as increased NO. The results support further study of CA and CD as potential treatments for ischemic heart disease.

Selective inhibition of PKCβ2 preserves cardiac function after myocardial infarction and is associated with improved angiogenesis of ischemic myocardium in diabetic rats

Activation of PKCβ2 induced by hyperglycemia contributes to impaired angiogenesis in endothelial cells. The purpose of the present study was to investigate whether PKCβ2 inhibition can attenuate the hyperglycemia-induced impaired angiogenesis of myocardium to improve cardiac function following myocardial infarction (MI) in diabetes. In vitro, human umbilical vein endothelial cells (HUVECs) were cultured in low glucose (L-G) (5.6 mmol/l) or high glucose (H-G) (33.3 mmol/l) medium in the presence or absence of LY333531 (LY333) (10 nmol/l), a selective PKCβ2 inhibitor. In vivo, with the use of an MI diabetic rat model, animals were randomized to receive LY333 (10 mg/kg/day) orally for 4 weeks after MI, or no treatment whatsoever. Treatment of HUVECs with LY333 prevented the H-G-induced decrease of tube formation, migration and proliferation. Furthermore, exposure of HUVECs to H-G activated PKCβ2 and decreased levels of phospho-Akt (p-Akt) and phospho-endothelial nitric oxide synthase (p-eNOS) expression, which was also prevented by LY333. Compared with MI rats without therapy, LY333-treated MI rats showed an increase in left ventricular ejection fraction (LVEF) and fractional shortening (FS), but a reduction in infarct size. Furthermore, treatment of rats with LY333 not only significantly increased the capillary density of ischemic myocardium, but also significantly elevated the levels of p-Akt and p-eNOS expression. We also observed a significant increase of VEGF expression in myocardium measured by immunostaining in MI and LY333 groups compared to sham group. Anti-CD31 immunostaining revealed that MI rats treated with LY333 exhibited increased density of capillaries compared with sham group rats. However, treatment of rats with LY333 did not result in significant increases in vascular endothelial growth factor (VEGF) expression at both the mRNA and protein levels in myocardium, and in the plasma level of VEGF compared with MI rats without therapy. Overall, these results suggest that inhibition of PKCβ2 may be a novel therapeutic approach for preserving cardiac function after MI, in part by improving impaired angiogenesis of myocardium in diabetes.

Electrophysiological studies of upregulated P2X7 receptors in rat superior cervical ganglia after myocardial ischemic injury

Myocardial ischemic injury activates cardiac sympathetic afferent fibers and elicits a sympathoexcitatory reflex by exciting sympathetic efferent action, with resultant augmentation of myocardial oxygen consumption, leading to a vicious cycle of exaggerating myocardial ischemia. P2X7 receptor participates in the neuronal functions and the neurological disorders. This study examined the role of P2X7 receptor of superior cervical ganglia (SCG) in sympathoexcitatory reflex. Our results showed that the expression of P2X7 receptor at both mRNA and protein in SCG was increased after myocardial ischemic injury. P2X7 receptor agonists at the same concentration activated much larger amplitudes of the currents in the SCG neurons of myocardial ischemic rats than those in control rats. P2X7 receptor antagonist (brilliant blue G, BBG) significantly inhibited P2X7 receptor agonist-activated currents in the SCG neurons. Excessive phosphorylation of MAPK ERK1/2 upon the activation of P2X7 receptor might be a mechanism mediating the signal transduction after myocardial ischemic injury. Therefore, the sensitized P2X7 receptor in SCG was involved in the nociceptive transmission of sympathoexcitatory reflex induced by myocardial ischemic injury.

Sensory-sympathetic coupling in superior cervical ganglia after myocardial ischemic injury facilitates sympathoexcitatory action via P2X7 receptor.

P2X receptors participate in cardiovascular regulation and disease. After myocardial ischemic injury, sensory-sympathetic coupling between rat cervical DRG nerves and superior cervical ganglia (SCG) facilitated sympathoexcitatory action via P2X7 receptor. The results showed that after myocardial ischemic injury, the systolic blood pressure, heart rate, serum cardiac enzymes, IL-6, and TNF-α were increased, while the levels of P2X7 mRNA and protein in SCG were also upregulated. However, these alterations diminished after treatment of myocardial ischemic (MI) rats with the P2X7 antagonist oxATP. After siRNA P2X7 in MI rats, the systolic blood pressure, heart rate, serum cardiac enzymes, the expression levels of the satellite glial cell (SGC) or P2X7 were significantly lower than those in MI group. The phosphorylation of ERK 1/2 in SCG participated in the molecular mechanism of the sympathoexcitatory action induced by the myocardial ischemic injury. Retrograde tracing test revealed the sprouting of CGRP or SP sensory nerves (the markers of sensory afferent fibers) from DRG to SCG neurons. The upregulated P2X7 receptor promoted the activation of SGCs in SCG, resulting in the formation of sensory-sympathetic coupling which facilitated the sympathoexcitatory action. P2X7 antagonist oxATP could inhibit the activation of SGCs and interrupt the formation of sensory-sympathetic coupling in SCG after the myocardial ischemic injury. Our findings may benefit the treatment of coronary heart disease and other cardiovascular diseases.